Human pancreatic lipase is a glycoprotein with a molecular mass of approximately 45,000 daltons. Lipase cleaves emulsified long-chain triglycerides to yield mono- and diglycerides, and fatty acids. It is known that cleavage of triglycerides by lipase only occurs at the oil/water interface of an oil in water emulsion. In other words, lipase activity is manifested only on the boundary surface of oil droplets existing as the dispersed phase in a continuous water phase. Lipase-induced cleavage of triglycerides is affected by substrate surface phenomena.
As an indication of both acute and chronic pancreatitis, pancreatic cancer, pancreatic injury, and miscellaneous abdominal disorders, a patient's serum and tissue lipase activity is a powerful diagnostic tool. Because of lipase's unique reaction mechanism, however, the diagnostic usefulness of lipase depends on the method used for its estimation.
Several methods for serum lipase assay are known. Older methods, such as the Cherry-Crandall method, required overnight sample incubation and time-consuming titration to a color-change end point. Because of the numerous steps involved, the procedures tended to suffer from poor reproducibility.
Variations on the Cherry-Crandall method have been reported; their primary limitation is the triglyceride emulsion. Because lipase activity varies with the overall surface area of the oil droplets in the emulsion, reproducible results were difficult to obtain. Even with the best efforts, the emulsions employed in these methods varied from batch to batch.
Another problem with early assay methods is their indifference to lipoprotein lipase interference. Particularly among patients receiving the drug heparin, falsely increased lipase values have resulted when lipoprotein lipase interference has not been accounted for.
Turbidimetric methods for lipase determination are known. In a typical turbidimetric assay, a sample of lipase-containing serum is mixed with a predetermined amount of a triglyceride-water emulsion, and the clarification of the turbidity of the emulsion is followed photometrically.
Although turbidimetric techniques have proven to be both more reliable and more convenient than older titrimetric serum lipase tests, the techniques suffer from certain drawbacks. The primary problem is due to non-uniformity of the size of the emulsified oil droplets. To permit spectrophotometry, the emulsion must be very dilute. Negative lipase values occasionally result, because increases in optical absorbance are obtained during the measurement phase of the test.
Attempts to improve the turbidimetric technique by solving the problem of droplet size non-uniformity are known. U.S. Pat. No. 4,343,897 to Neumann et al. describes a dry reagent for the turbidimetric determination of lipase, which forms an emulsion when water is added thereto. The dry reagent is prepared by lyophilization of an emulsion produced by conventional methods. In addition to the expense of lyophilization, the reagent suffers from the inherent drawback of potential human error in the reconstitution of the emulsion.
Accordingly, a need exists for a reagent for the determination of lipase that gives consistently uniform results, is easily prepared in toto in the factory, and is ready to use in the field as a single system reagent. The present invention fulfills this need.